The present invention is concerned with an improved apparatus and method for detecting, identifying and testing microorganisms isolated from liquids, particularly but not necessarily blood or other body fluids.
One particularly important area of use for the present invention is in the diagnosis of bacteremia. Bacteremia is a clinically significant, potentially life-threatening event. Despite the availability of a number of effective antimicrobial agents, the rate of mortality associated with bacteremia remains high. Recovery from bacteremia is increased when appropriate antimicrobial therapy is instituted early in the course of infection. Therefore, prompt detection, identification and susceptibility testing of microorganisms isolated from blood is imperative.
Procedures for diagnosing bacteremia require rapid isolation of the infecting organism. Culturing blood in a growth medium using commercially available blood culture bottles is the standard isolation technique. However, this technique has several disadvantages. Thus, for example, detection can be affected by the type of medium, the density of bacteria in blood, the time of collection, the volume of blood, the host's immune response, laboratory practices and other factors. In addition, several hours or days may be required to obtain results, depending on the bacteria's growth characteristics.
All of the above noted factors cause delays in obtaining the required diagnosis. Additionally, contamination is a real problem and the indicated test procedures can lead to inaccuracies in the diagnosis itself.
Much time and effort have been spent over many years in the search for more effective alternative methods to speed the isolation and identification of infecting bacteria. Various techniques for concentrating and isolating microorganisms from blood have been explored with little success. Sedimentation-filtration, lysis-filtration with measure of impedance and, more recently, charcoal hemoperfusion of the patient's blood have all been utilized. However, each method has suffered from one or more significant drawbacks. Typical problems include clogging of filters with serum and cells, toxicity of lysing solutions, contamination and decreased sensitivity. As a consequence, there has only been limited acceptance of previously available systems by clinical microbiology laboratories.
The present invention offer an improved method and apparatus for detecting and identifying microorganisms in blood or other body fluids or the like which avoid or substantially reduce the number of problems encountered with previously available approaches. A number of the advantages of the present method and apparatus are detailed hereinafter. Broadly speaking, however, the invention provides a relatively quick, simple and accurate bacterial culture system for isolating and identifying microorganisms, particularly those isolated from the blood or other body fluids.
The invention is based, to a significant extent, on a special combination and adaptation of centrifugation, filtration and growth media techniques which cooperate to expedite the recovery and identification of microorganisms from blood or the equivalent. Typically, according to the invention, fresh whole human blood is mixed with a fluid density gradient in a specially designed collection tube generally similar to the conventional vacutainer. The tube is centrifuged in standard manner for a suitable period of time, e.g. 30 minutes or so, to separate erythrocytes by sedimentation. The remaining fluid (plasma, leukocytes and fluid gradient) is then vacuum drawn from the tube through a special membrane filter after which a media cup containing a selected growth or culture medium is attached to the base of the filter to promote bacterial growth. The collected microorganisms are then detected on the filter within an appropriate culture period, e.g. 18-24 hours. The collection tube and filter unit are so designed that the tube may be sealed into the filter unit for application of the filtration vacuum before the tube contents are open to the vacuum whereby possible contamination of the fluid is kept to a minimum. The media cup likewise has a special sealing connection to the filter unit and the filter membrane is tightly sealed within the filter unit itself to avoid contamination. Additionally, the media cup is positioned close to, i.e. just below and functionally in contact with, the filter membrane so that the microorganisms collected on the filter membrane can be cultivated essentially "in situ".
The system of the invention is effective and sensitive for the recovery and detection of organisms in blood fluids or the like even when the organisms are present in very small amounts. Tests conducted with seeded or inoculated human blood, using concentrations of inocular in the range of from 3.0.+-.0.7 to 18.0.+-.3.6 organisms per ml have been successfully conducted and indicate no statistically significant differences detectable between the number of microorganisms recovered by the present system and by the conventional culture of the original inoculum.
The invention is useful in detecting any type of known micro-organism larger than viruses which may be found in the analysis of blood, other body fluid or the like. Typical of such microorganisms are Candida albicans, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Listeria monocytogenes, Neisseria meningitidis, Pseudomonas aeruginosa, Staphyllococcus aurias, Streptococcus faecalis, and Streptococcus pneumoniae. These microorganisms have been detected on the filter membrane, using the method and apparatus of the invention, within 18 hours after filtration. Detection times will vary depending on various factors, for example, the amount of micro-organisms present. Thus, in tests using blood inoculated with the above listed micro-organisms, it has been found that detection time is decreased by 3 to 4 hours with each four to five-fold increase in inoculum. Similar results have been obtained with anaerobic organisms such as Bacteroides fragilis, Fusobacterium nucleatum, Peptococcus asaccharolyticus, Peptostreptococcus micros and Veilonella parvula.
It will be recognized by those in the art that the combination of centrifugation and filtration procedures contemplated for use herein constitutes a departure from conventional blood culture systems. Filtration procedures have not been well accepted because of the amount of manipulations generally required to process blood. The present system, however, requires a minimum of such manipulations and offers major advantages over previous techniques involving centrifugation and filtration. For example, no lysing agents, multiple filters, dilutions, sophisticated equipment or inordinate centrifugation speeds are required or involved. Clogging of filters because of contamination with leukocytes and erythrocytes is eliminated and a minimum amount of time, e.g. less than 30 seconds, is required to filter the entire gradient. In addition, as noted earlier, the blood collecting tube, filter and media are designed to prevent contamination, the overall system being effectively sealed.
The invention is further described by reference to the accompanying drawings wherein: